mmg_233_2013_genetics_genomicswikiaorg-20200214-history
FISH (fluorescence in situ hybridization)
Fish is a technique developed in the 1980's that allows scientists to observe the structure and function of a cell. The technique uses florescent probes to detect the presents or absents of DNA sequences on chromosomes under a Fluorescence microscope [1.] Origin of FISH FISH was developed in 1980 to replace radiolabled probes that were used to detect specific molecular identifies of a cell. Radiolabled probes were not ideal because isotopes decay, their resolution is limited, the exposure time is long, and they are costly and hazardous. The development of FISH was safer, faster, and had a higher resolution then the radioactive probes [2.] The first time FISH was used a fluorophore was placed on the 3' end of an RNA stran d and it then attached to a specific sequence of DNA. However, the probe was not overly specific due to the low copy of nucleic acids so to increase the single output of the fluorophore secondary probes had to be used to detect the DNA and RNA targets. Later scientist were able to better label the single strand DNA and this allowed proper chemical preparation of the probes that carried the florescent molecules used for detection [2.] Development of FISH In the beginning the development of FISH focused on expansion of the types of probes used and the number of targets they can detect. The future of FISH looks to expend the number of subjects used in an investigation. At first the specimen thickness was limited to a 1-2 mm thickness by the fluorescence microscopy now specimen used can be 15 mm thick. Also to reduce operator error advances have been in the mechanics that deliver, image, and analyze the specimen. Scientists look forward to using FISH with other technologies that use fluorescent fusion proteins to simultaneously monitor proteins and the nucleic acids of interest [2. ] FISH Step by Step Probes were first derived from fragments of DNA that were isolated, derived, and purified during the Human Genome Project. The fragments were then placed into plasmids to be preserved and then later replicated in bacterial colonies. These colonies are called bacterial artificial chromosomes and are used in FISH because they can be easily grown, extracted, and labeled with a fluorophore. After the probe is labeled with the fluorophore using nick translation or PCR the chromosome of interest must be prepared. To prepare the chromosome it is arrested in interphase or metaphase and then fixed to a substrate. The probe is then attached to the chromosome and the reporter gene can signal where the gene of interest is on the chromosome [1.] For a detailed step by step experiment you can refer to a FISH protocol supplied by the company Invitrogen. A company called AneuVysion provides multicolor FISH probes for prenatal genetics. Uses for FISH Scientists most commonly use FISH probes in three different situations. The first FISH probe is the locus specific probe, which binds to a specific region on a chromosome. This is helpful if a scientist want to know what chromosome a specific gene is located on. The second is generated from the middle repeat portion of the chromosome and is called the centromeric repeat probe. This is used to determine if an individual contains the correct number of chromosomes. When used in combination with the locus specific probes it can determine if an individual has genetic material missing and from which chromosome it is missing from. Finally the third most common use is the whole chromosome probe. This uses multiple smaller probes that bind to different regions of a whole chromosome. Each chromosome can then have its own color and a spectral karyotype is produced. This is helpful to observe chromosomal abnormalities like when one chromosome has a piece of another chromosome attached to it [3.] Examples of FISH in Genetics FISH can be useful in pre-implantation genetic diagnosis to detect chromosome imbalances like chromosomal translocations which can result in cancers and down syndrome, and X-linked diseases like hemophilia A, and Duchenne muscular dystrophy. This is done by removing a single cell from an embryo 3 days after fertilization. The FISH probes are then attached to the single cells chromosomes to detect the chromosomal imbalances [5. ] References 1. Wikipedia (2013) Link: http://en.wikipedia.org/wiki/Fluorescence_in_situ_hybridization 2. Fluorescence in situ hybridization: past, present and future (2003) Link: http://jcs.biologists.org/content/116/14/2833.full 3. National Human Genome Research Institute (2011) Link: http://www.genome.gov/100002 4. Protocol by Invitrogen ( 2011) Link: http://probes.invitrogen.com/media/pis/mp20912.pdf 5. FISH for pre-implantation genetic diagnosis (2010) Link: http://www.ncbi.nlm.nih.gov/pubmed/20809319